EP2537601B1 - Trousse et procédé de détection de biofilms - Google Patents

Trousse et procédé de détection de biofilms Download PDF

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Publication number
EP2537601B1
EP2537601B1 EP11171360.8A EP11171360A EP2537601B1 EP 2537601 B1 EP2537601 B1 EP 2537601B1 EP 11171360 A EP11171360 A EP 11171360A EP 2537601 B1 EP2537601 B1 EP 2537601B1
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EP
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Prior art keywords
biofilms
biofilm
detecting
vol
solution
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EP11171360.8A
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German (de)
English (en)
French (fr)
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EP2537601A1 (fr
Inventor
Gauthier Boels
Gordon Blackman
Almudena Calabozo
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Realco SA
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Realco SA
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Priority to DK11171360.8T priority Critical patent/DK2537601T3/da
Application filed by Realco SA filed Critical Realco SA
Priority to PL11171360T priority patent/PL2537601T3/pl
Priority to EP11171360.8A priority patent/EP2537601B1/fr
Priority to PT111713608T priority patent/PT2537601T/pt
Priority to ES11171360T priority patent/ES2873233T3/es
Priority to US14/126,320 priority patent/US20140113326A1/en
Priority to PCT/EP2012/062086 priority patent/WO2012175671A1/fr
Priority to CA2836868A priority patent/CA2836868C/en
Publication of EP2537601A1 publication Critical patent/EP2537601A1/fr
Priority to US14/601,158 priority patent/US10683529B2/en
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Publication of EP2537601B1 publication Critical patent/EP2537601B1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/22—Testing for sterility conditions
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements

Definitions

  • the present invention relates to a method and to a kit for detecting biofilms, compatible with the food industry.
  • Hygiene is becoming increasingly important in the food industry, in hospitals, in water purification and desalination installations, in the treatment of process water, and in particular in water used in cooling towers and in the necessities of everyday life, for example contact lenses. It is frequently observed that, during the circulation of water or substances rich in nutrient material on a support, microorganisms circulating freely in the water or in the nutrient material can adhere to the surface. These microorganisms can then develop an adhesive extracellular matrix composed of polymeric substances.
  • Biofilms A community of microorganisms adhered to a surface and embedded in such a matrix is called a biofilm. Usually these biofilms are composed of bacteria and organic polymers produced by them. Biofilms are now developing on all types of support such as food conveyor belts in the food industry, on supports intended to maintain a nutrient substance or in any case organic for any step such as for example the hooks of keeping meat and the like. Biofilms also develop on the worktops of the horeca, on cleaning installations such as drainers, taps, splashbacks, dishwashers, etc.
  • biofilms are also observed in medical facilities such as operating theaters and others.
  • medical devices where liquid phases are present (body fluids, aqueous phase for cleaning after use, etc.).
  • biofilms such as the paper industry, the sludge treatment industry and any other industry in which plant or organic solids are digested in the presence of water or water. a similar aqueous solution.
  • this matrix is very resistant, and can constitute a barrier for agents which would act against microorganisms.
  • Conventional treatments based on sodium hydroxide and / or comprising different biocides do not act in a sufficiently efficient manner because they do not penetrate the biofilm over its entire thickness or are inhibited by certain molecules making up this matrix. The treatment is then only partially effective on the upper surface of the biofilm. In addition, the latter can also trap other microorganisms, pathogens in particular, than that which was installed initially. Therefore, a simple cleaning of the installations is generally not sufficient and requires a more specific treatment of the areas contaminated by biofilms.
  • biofilms Unfortunately, the specific treatments of biofilms are more restrictive than a simple conventional cleaning step and therefore require that the contaminated areas be easily identified. If in some cases, said extracellular matrix is easily identified when it is highly developed, in other cases, the biofilm develops insidiously in the installations and its presence is only detected during the analysis of the quality of the product. finished.
  • Biofilms are inevitably formed (in view of the richness of the surrounding environment).
  • Biofilms exhibit cyclic growth activity including a growth phase lasting which occurs the accumulation of microorganisms and a detachment phase during which entire pieces of biofilms are detached by erosion and under the effect of their own weight.
  • a growth phase lasting which occurs the accumulation of microorganisms
  • a detachment phase during which entire pieces of biofilms are detached by erosion and under the effect of their own weight.
  • this type of detection makes it possible to know that a biofilm is present somewhere, but does not make it possible to locate it precisely.
  • the production is not stopped and when the biofilm is in the breaking phase, the produced batches are contaminated and discarded until the level of contamination by the microorganisms of the food produced is again acceptable in the market. in view of the standards in force.
  • the document JP 2005210997 discloses a coloring composition for detecting biofilms in the food industry containing rice red (monascin) which is sprayed onto surfaces likely to contain biofilms.
  • This composition comprises 92% water, 4% dye and 4% ethanol. Although compatible with the food industry, this composition requires a high dye content, is not very compatible with an alkaline medium (used to sanitize installations in a conventional manner) and has a color that is difficult to observe (probably justifying the high dye content).
  • the document EP 1491505 discloses for its part a method for measuring the formation of biofilm in aqueous systems. This method includes a first step of placing sample coupons in the aqueous system. It is then necessary to wait for a biofilm to develop before recovering the coupons. The coupons are then stained with a dye, rinsed and analyzed by photometry or by comparison with a calibration card.
  • the detection method according to this document has several drawbacks, in particular the fact that it reveals certain uncertainties and that it is really long. Indeed, the placement of coupons must be carried out in areas in which the presence of biofilms is suspected (suggesting that they have already been detected before) and wait for the development of the latter. Although the bacteria are fast growing, it is by no means excluded that a biofilm will not grow on the coupon while the facility is covered with biofilms or the time during which the coupons are placed in the installation is not adequate for a biofilm to grow on the coupon. Bacteria always exhibiting a higher affinity for the extracellular matrix already formed on the installation to be treated than for the smooth surface of the sample coupon.
  • the coupons are rinsed with azide to eliminate bacterial growth.
  • the staining solution may be an aqueous solution of safranin, coomassie blue, crystal violet, ruthenium red, or erythionine.
  • the rinsing solution is a solution containing azide and the coupons are finally washed with DMSO after drying.
  • the aim of the invention is to overcome the drawbacks of the state of the art by providing a method and a kit for detecting biofilms making it possible to detect on any type of surface the presence of biofilms, which are particularly versatile, that is to say. say usable for any type of application, including in the food industry where from a health point of view, biofilms must be detected quickly and precisely and efficiently, that is to say where the detection of false negative and false positive is limited or nonexistent.
  • the dye dilution phase is compatible with any type of application, but also with the food industry, which allows it to be applied in all types of industrial installations and the dye used is easily available on the market and particularly visible.
  • the combination of the staining solution according to the invention and the cleaning solution makes it possible to reduce the detection of false positives and improves the selectivity of the detection with respect to existing detections.
  • the polysaccharides of bacteria may not be detected since the bacteria are confined in this polymer matrix and therefore are not accessible for staining. , especially if the dye has a short dwell time.
  • the kit according to the invention has a speed of detection and a very advantageous specificity because it targets the glycoproteins of the extracellular matrix of the biofilm and therefore reduces the presence of false negatives or false positives and targets the glycoproteins. very accessible by the dye and not very present in food or other residues. The biofilm is therefore quickly and selectively detected.
  • the detection kit according to the invention simply comprises a first staining solution to be sprayed and a cleaning solution to be sprayed subsequently, the dwell times of which are short (less than 15 minutes). This means that generally, in less than an hour, preferably in less than half an hour, the biofilms are detected and located precisely.
  • the practical aspect of the kit according to the invention is particularly advantageous in that it does not require an expensive installation to detect the biofilm, the user's eye is sufficient, which is due to the particular choice of the dye, easily identifiable with the naked eye, different from the colors generally found in all types of industries dealing with plant or organic matter (few blue foods, unlike red). A simple spraying of the first solution followed by the second is sufficient.
  • the detection kit according to the invention further comprises a bleaching composition, compatible with the food industry, which then makes it possible to remove the blue color from the installation if necessary.
  • a bleaching composition compatible with the food industry, which then makes it possible to remove the blue color from the installation if necessary.
  • the materials used are porous, such as the rubbers of conveyor belts.
  • a bleaching solution will be used. It is in fact not advantageous for the blue color to persist after use because this could be detrimental to the subsequent detection steps.
  • the blue color of the biofilm is of course eliminated with the biofilm.
  • said bleaching composition is a solid phase of an oxidizing agent which can therefore simply be spread over the treated area, preferably wetted beforehand to promote activation of the oxidizing agent in water.
  • the presence of the bleaching composition in the form of a solid phase is advantageous for the preservation of the oxidizing character of the bleaching composition over time.
  • said bleaching composition is an aqueous solution of an oxidizing agent, which allows it to be easily applied to the area to be treated by simple spraying, especially when it is vertical.
  • said oxidizing agent is chosen from the group consisting of sodium percarbonate, sodium hypochlorite, hydrogen peroxide, perborates, persulphates or else. peroxides or and their mixtures and their derivatives, such as, for example, urea percarbamate.
  • said dilution phase comprises from 40 to 50%, more preferably approximately 45% by volume of ethanol, in particular absolute ethanol, relative to the final volume of said dilution phase, from 8 to 12% , more particularly approximately 10% by volume of acetic acid, in particular glacial acetic acid, relative to the final volume of said dilution phase, and from 40 to 50%, more preferably approximately 45% by volume of water, relative to the final volume of said dilution phase.
  • this dilution phase only comprises substances that are ingestible and compatible with the food industry, which can be easily obtained and inexpensively.
  • the method according to the present invention is fast, inexpensive in terms of time and equipment to use, efficient by its selectivity and its speed of detection and compatible with the food industry, therefore versatile for all types of food. industry.
  • one or more of the steps chosen from the group of the step of vaporization of said coloring solution, of vaporization of said cleaning solution and of detection of said biofilm is preceded by a step of rinsing with water, which makes it possible to eliminate the surplus of dye, on the zone to be treated or diluted in the cleaning solution, which makes it possible to improve the selectivity of the method for detecting biofilms according to the invention.
  • the method further comprises a bleaching step said residual areas stained with coomassie blue by application of a bleaching composition.
  • said bleaching step is carried out by applying a solid bleaching composition, in particular a pulverulent composition of an oxidizing agent, to the surface to be treated which is wet beforehand to promote activation of the oxidizing agent.
  • a solid bleaching composition in particular a pulverulent composition of an oxidizing agent
  • said bleaching step is carried out by applying a liquid bleaching composition of an oxidizing agent.
  • said predetermined period of time is between 3 and 15 minutes, preferably between 4 and 10 minutes and is generally about 5 minutes. This of course makes it possible to rapidly detect the biofilm, but also reflects the efficiency of the method according to the invention which can be implemented rapidly and its selectivity (the substance to be detected is actually targeted and quickly detected).
  • said surface liable to be contaminated by a biofilm is an open surface of an installation, in particular of an installation in the food industry. It is in fact preferable that the surface on which a biofilm is to be detected by the method according to the invention is a surface visible to the eye, this is what is generally called an open surface.
  • the method according to the invention can also be applied to surfaces that are less accessible or not visible, but they will undoubtedly have to be dismantled for the detection step, for example, tubes can be treated, for example by circulation. , but they will most likely need to be opened to detect the result.
  • the method can also resort to the use of coupons placed in closed facilities. Once collected, the coupons are then treated like any open surface within the meaning of the present invention.
  • the present invention also relates to a use of a biofilm staining solution consisting of a dye which is Coomassie blue in solution in a dilution phase compatible with the food industry, comprising from 35 to 55% by volume of 'ethanol, from 7 to 13% by volume of acetic acid and from 35 to 55% by volume of water, relative to the final volume of said dilution phase, and a ready-to-spray cleaning solution comprising said phase of dilution, on a surface liable to be contaminated by a biofilm, said surface being an open surface, in particular in an installation of the food industry, in order to detect and locate said biofilm.
  • a biofilm staining solution consisting of a dye which is Coomassie blue in solution in a dilution phase compatible with the food industry, comprising from 35 to 55% by volume of 'ethanol, from 7 to 13% by volume of acetic acid and from 35 to 55% by volume of water, relative to the final volume of said dilution phase, and
  • the figure 1 illustrates the biofilm detection kit 1 comprising a staining solution 2 of biofilms containing coomassie blue in solution in a dilution phase compatible with the food industry.
  • the dye can obviously, depending on the required applications possibly comprise another dye in mixture, but will include in any case, coomassie blue for the purpose of detecting biofilms.
  • the dilution phase of the coloring solution 2 comprises, in this advantageous embodiment illustrated 45% by volume of absolute ethanol, 10% by volume of glacial acetic acid and 45% by volume of water relative to the final volume. of said dilution phase.
  • the detection kit 1 further comprises a cleaning solution 3 advantageously made up of said dilution phase. This of course makes it possible to dilute the dye which is not bound to the proteins of the biofilm.
  • the biofilm detection kit 1 also advantageously comprises a bleaching composition 4, compatible with the food industry.
  • the bleaching composition 4 is a solid phase of an oxidizing agent composed of sodium percarbonate.
  • the bleaching composition 4 is an aqueous solution of an oxidizing agent, such as for example a solution of sodium hypochlorite or hydrogen peroxide.
  • the surface to be treated to detect whether a biofilm is present or not for example an open surface from the food industry is rinsed and then sprayed with water. using the coloring solution 2.
  • the coloring is left to act for approximately 5 minutes and the surface to be treated is then rinsed to remove the excess coloring solution therefrom.
  • the amount of coloring solution used will be approximately 6.5 ⁇ l / cm 2 of surface to be treated, ie 65.2 ml / m 2 .
  • the surface to be treated is then, according to the recommendations, sprayed with cleaning solution 3 which is left to act for approximately 5 minutes.
  • the cleaning solution advantageously consists of the dye dilution phase of the staining solution 2 of the biofilm detection kit according to the present invention.
  • the cleaning solution therefore eliminates any traces of excess coomassie blue which does not adhere to the biofilm by the effect of dilution in the dilution phase.
  • the amount of coloring solution used will be approximately 21.7 ⁇ l / cm 2 of surface to be treated, ie 217 ml / m 2 .
  • the surface is then rinsed with water to remove the residual traces of coomassie blue diluted in cleaning solution 3 and the surface to be treated is then left to stand for 5 minutes.
  • blue coloration persists on the surface to be treated, it indicates the presence of a biofilm and therefore allows its rapid detection in approximately less than 20 minutes.
  • the bleaching composition in the form of a powdered reagent in the kit according to the invention is distributed over the wet surface to be treated in order to remove the blue coloration from the surface to be treated.
  • the coupons are then placed in the colored solution for 30 minutes containing approximately 0.25 g of Coomassie blue R250 in a dilution phase comprising 45 ml of pure ethanol, 45 ml of distilled water and 10 ml of glacial acetic acid. .
  • the coupons are then immersed in 100 ml of cleaning solution consisting of approximately the same dilution phase. Then the coupons were allowed to air dry.
  • the biofilm detection kit according to the present invention makes it possible to detect biofilms with high specificity.
  • EXAMPLE 2 Industrial example in a food industry.
  • the coloring solution of the kit according to the invention was sprayed on all the devices of these rooms such as hooks, head puller, blades, stainless steel containers, cups, etc. and left to act for 5 minutes.
  • the coloring solution comprising 0.1 g of coomassie blue in 100 ml of dilution phase comprising 45 ml of water, 45 ml of absolute ethanol and 10 ml of glacial acetic acid.
  • the surfaces of the elements tested were rinsed with water to remove excess dye and then sprayed with the cleaning solution comprising said dilution phase.
  • the cleaning solution was left to act for 5 minutes and the elements tested for the detection of biofilms were rinsed and mechanically rubbed to provide a slight mechanical action and to remove any residual stains and / or false positives. The results were then observed. It is very easy to see that the problems of persistent contamination on certain surfaces are caused by the presence of biofilm, such as for example on the elements tested such as plucking hooks, blades, cups, containers and head pullers. Only the head pullers are illustrated on the figure 2 although the other elements are also contaminated. Despite the quality of cleaning performed by the company, some devices are sometimes severely contaminated by a biofilm. This biofilm is the source of non-compliant microbiological results
  • Example 1 The 5 sets of different coupons of Example 1 according to the invention were covered with the same different organic materials frequently used in the food industry except for the fact that for milk, two tests were carried out, the dilution of milk applied was 00 times as in Example 1, but also 20 times.
  • the coupons are then placed in the colored solution containing approximately 3 g of rice red (monascin) in 100 ml of a dilution phase comprising water and ethanol in a 1/1 ratio for 15 minutes, then for rinsing, 10 minutes in distilled water.
  • a dilution phase comprising water and ethanol in a 1/1 ratio for 15 minutes, then for rinsing, 10 minutes in distilled water.
  • Example 1 The 5 different sets of coupons of Example 1 were covered with the same different organic materials frequently used in the food industry except for the fact that for milk, two tests were carried out, the dilution of the milk applied was 00 times as in Example 1, but also 20 times.
  • the coupons are then placed in the colored solution containing approximately 36 g / l of safranin in a dilution phase comprising demineralized water, methyl ethyl ketone and ethanol for 15 minutes, then for rinsing, 10 minutes in water. demineralized water.

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EP11171360.8A 2011-06-24 2011-06-24 Trousse et procédé de détection de biofilms Active EP2537601B1 (fr)

Priority Applications (9)

Application Number Priority Date Filing Date Title
PL11171360T PL2537601T3 (pl) 2011-06-24 2011-06-24 Zestaw i sposób wykrywania biofilmu
EP11171360.8A EP2537601B1 (fr) 2011-06-24 2011-06-24 Trousse et procédé de détection de biofilms
PT111713608T PT2537601T (pt) 2011-06-24 2011-06-24 Kit e método para deteção de biofilme
ES11171360T ES2873233T3 (es) 2011-06-24 2011-06-24 Kit y procedimiento de detección de biopelículas
DK11171360.8T DK2537601T3 (da) 2011-06-24 2011-06-24 Biofilmdetekteringskit og fremgangsmåde
PCT/EP2012/062086 WO2012175671A1 (fr) 2011-06-24 2012-06-22 Trousse de detection de biofilms
US14/126,320 US20140113326A1 (en) 2011-06-24 2012-06-22 Kit for detecting biofilms
CA2836868A CA2836868C (en) 2011-06-24 2012-06-22 Kit for detecting biofilms
US14/601,158 US10683529B2 (en) 2011-06-24 2015-01-20 Kit for detecting biofilms

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
EP11171360.8A EP2537601B1 (fr) 2011-06-24 2011-06-24 Trousse et procédé de détection de biofilms

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EP2537601A1 EP2537601A1 (fr) 2012-12-26
EP2537601B1 true EP2537601B1 (fr) 2021-04-07

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US (2) US20140113326A1 (pl)
EP (1) EP2537601B1 (pl)
CA (1) CA2836868C (pl)
DK (1) DK2537601T3 (pl)
ES (1) ES2873233T3 (pl)
PL (1) PL2537601T3 (pl)
PT (1) PT2537601T (pl)
WO (1) WO2012175671A1 (pl)

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BONNIE MARCHANT ET AL: "Developing Fingerprints in Blood: A Comparison of Several Chemical Techniques", vol. 57, no. 1, 1 January 2007 (2007-01-01), pages 76 - 93, XP009511210, ISSN: 0895-173X, Retrieved from the Internet <URL:https://www.ncjrs.gov/App/Publications/abstract.aspx?ID=238743> *

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PT2537601T (pt) 2021-05-28
DK2537601T3 (da) 2021-05-10
US20140113326A1 (en) 2014-04-24
PL2537601T3 (pl) 2021-08-02
US20150132796A1 (en) 2015-05-14
EP2537601A1 (fr) 2012-12-26
CA2836868A1 (en) 2012-12-27
WO2012175671A1 (fr) 2012-12-27
ES2873233T3 (es) 2021-11-03
CA2836868C (en) 2022-02-08

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